DETECTION OF PYRIMIDINE DIMERS AND MONOADDUCTSINDUCED BY 7-METHYLPYRIDO(3,4-c) PSORALEN AND UVA IN CHINESE HAMSTER V79 CELLS BY ENZYMATIC CLEAVAGE AND HIGH-PRESSURE LIQUID CHROMATOGRAPHY

Abstract The photochemotherapeutically active psoralen derivative 7-methylpyrido(3,4-c) psoralen (MePyPs) has been recently shown to be able to photoinduce monoadducts of the C₄-cycloaddition type as well as pyrimidine dimers in DNA in vitro . In the present study, we report on the induction of these two types of photolesions in mammalian cells in culture. The MePyPs photocycloadducts were quantified in V79 Chinese hamster cells after treatment with MePyPs plus UVA following enzymatic hydrolysis of the DNA by DNase I, S1 nuclease and acidic phosphatase treatments. Concomitantly induced pyrimidine dimers were determined by two methods, high-pressure liquid chromatography and alkaline gel electrophoresis after dimer-specific endonucleolytic cleavage. The results show that, in Chinese hamster cells treated with MePyPs plus UVA, the yield of pyrimidine dimers is approximately 5-10% that of MePyPs-DNA photocycloadducts. Because psoralen monoadditions to DNA alone are generally not considered as being very phototoxic, a synergistic interaction of monoadditions with pyrimidine dimers may be expected to occur in order to explain the high photobiological effectiveness of this psoralen derivative.     

Micellar electrokinetic capillary chromatography and data alignment analysis: a new tool in urine profiling

The complex nature of biofluids demands efficient, sensitive and high-resolution analytical methodologies to examine how the 'metabolic fingerprint' changes during disease. This paper describes how sulphated beta-cyclodextrin-modified micellar electrokinetic capillary chromatography (SbetaCD-MECC) has been combined with data alignment analysis and may prove a useful new tool in urine profiling, allowing for separation of over 80 urinary analytes in under 25 min. The optimised and validated SbetaCD-MECC methodology combined with data alignment analysis provides rapid identification of 'mismatches' between urine profiles which are not easily detected with the naked eye as well as a 'similarity score' which indicates the total sum of differences between one profile and another. The combination of SbetaCD-MECC with data alignment software should prove a useful alternative tool in metabonomic studies for rapid comparison of urine profiles.     

Affinity assays for detection of cellular communication and biomarkers

The biological events occurring in the body are complex and challenging to decode. The expression, production, secretion and interaction of proteins, peptides and small molecules often occur in a fast manner and at low concentrations. Methods used to quantify these events must be rapid, selective, sensitive and robust. In recent years, new variations of affinity methodologies have been developed to facilitate quantitation of these biomolecules. This review will focus on selected affinity techniques that have described multi-analyte measurement, high sensitivity techniques, or the application of new affinity reagents applied to conventional technologies to measure analytes involved in cell communication and biomarkers produced in specific disease states.     

New technologies in affinity assays to explore biological communication

The language that cells use to communicate consists of the small molecules, peptides, and proteins that are released into the extracellular environment. To decipher this language, analytical assays are needed that have high selectivity, high sensitivity, and fast temporal resolution. Affinity assays are a group of analytical methodologies that are adept at studying this communication. In this overview, we highlight several examples from the literature on various types of affinity assays used in different platforms to monitor biological communication of peptides and proteins.     

Simultaneous capillary electrophoresis competitive immunoassay for insulin, glucagon, and islet amyloid polypeptide secretion from mouse islets of Langerhans

A capillary electrophoresis competitive immunoassay was developed for the simultaneous quantitation of insulin, glucagon, and islet amyloid polypeptide (IAPP) secretion from islets of Langerhans. Separation buffers and conditions were optimized for the resolution of fluorescein isothiocyanate (FITC)-labeled glucagon and IAPP immunoassay reagents, which were excited with the 488 nm line of an Ar(+) laser and detected at 520 nm with a photomultiplier tube (PMT). Cy5-labeled insulin immunoassay reagents were excited by a 635 nm laser diode module and detected at 700 nm with a separate PMT. Optimum resolution was achieved with a 20mM carbonate separation buffer at pH 9.0 using a 20 cm effective separation length with an electric field of 500 V/cm. Limits of detection for insulin, glucagon, and IAPP were 2, 3, and 3 nM, respectively. This method was used to monitor the simultaneous secretion of these peptides from as few as 14 islets after incubation in 4, 11, and 20 mM glucose for 6h. For insulin and IAPP, a statistically significant increase in secretion levels was observed, while glucagon levels were significantly reduced in the 4 and 11 mM glucose conditions. To further demonstrate the utility of the assay, the Ca(2+)-dependent secretion of these peptides was demonstrated which agreed with published reports. The ability to examine the secretion of multiple peptides may allow for the determination of regulation of secretory processes within islets of Langerhans.     

Photocatalyzed sulfide oxygenation with water as the unique oxygen atom source

In our research program aiming to develop new ruthenium-based polypyridine catalysts for oxidation we were interested in combining a photosensitizer and a catalytic fragment within the same complex to achieve catalytic light-driven oxidation. To respond to the lack of such conjugates, we report here a new catalytic system capable of using light to activate water molecules in order to perform selective sulfide oxygenation into sulfoxide via an oxygen atom transfer from H(2)O to the substrate with a TON of up to 197 ± 6. On the basis of electrochemical and photophysical studies, a proton-coupled electron-transfer process yielding to an oxidant Ru(IV)-oxo species was proposed. In particular, the synergistic effect between both partners in the dyad yielding a more efficient catalyst compared to the bimolecular system is highlighted.     

Toxicological classification of urine samples using pattern recognition techniques and capillary electrophoresis

In toxicology, hazardous substances detected in organisms may often lead to different pathological conditions depending on the type of exposure and level of dosage; hence, further analysis on this can suggest the best cure. Urine profiling may serve the purpose because samples typically contain hundreds of compounds representing an effective metabolic fingerprint. This paper proposes a pattern recognition procedure for determining the type of cadmium dosage, acute or chronic, administrated to laboratory rats, where urinary profiles are detected using capillary electrophoresis. The procedure is based on the composition of a sample data matrix consisting of areas of common peaks, with appropriate pre-processing aimed at reducing the lack of reproducibility and enhancing the potential contribution of low-level metabolites in discrimination. The matrix is then used for pattern recognition including principal components analysis, cluster analysis, discriminant analysis and support vector machines. Attention is particularly focussed on the last of these techniques, because of its novelty and some attractive features such as its suitability to work with datasets that are small and/or have low samples/variable ratios. The type of cadmium administration is detected as a relevant feature that contributes to the structure of the sample matrix, and samples are classified according to the class membership, with discriminant analysis and support vector machines performing complementarily on a training and on a test set.     

SELECTIVE DNA THYMINE DIMERIZATION DURING UVA IRRADIATION IN THE PRESENCE OF A SATURATED PYRIDOPSORALEN

Abstract— It has been recently shown that UVA (320–400 nm) irradiation of DNA in the presence of pyridopsoralens induces the formation of thymine cyclobutane dimers in addition to monoadducts. In this work, we measured the potency of a saturated pyridopsoralen to photosensitize DNA, despite its inability to covalently attach to DNA. First, from spectroscopic fluorescence measurements, we have shown that both analogs, saturated and unsaturated pyridopsoralens, namely 4',5'-dihydro-7-methyl-pyrido[3,4-clpsoralen (DH-MePyPs) and 7-methylpyrido[3,4-c]psoralen, exhibit a similar global affinity for DNA. Secondly, we demonstrated, by footprinting experiments, that exposure of a DNA sequence to 365 nm UV radiation in the presence of DH-MePyPs results in selective cyclobutane thymine dimerization. Thymines located in the immediate proximity of the 5'-TA-3' step are exclusively affected and the frequency of this photoprocess depends on flanking sequences. We thus probe a selective thymine dimer photosensitizer. Results are discussed in terms of drug affinity and physical properties of the helix at the binding site.     

Validation and Further Optimisation of a Cyclodextrin-Modified Micellar Electrokinetic Capillary Chromatography Method for Urine Profiling

Metabonomic studies require efficient and high-resolution analytical probes to monitor changes in the ‘metabolic fingerprint’. The advantageous characteristics of Capillary Electrophoresis, enabling highly efficient separations of diverse components present in minute sample volumes, may therefore prove a useful tool in biofluid analysis. This paper describes the optimisation and validation of a sulphated β-cyclodextrin-modified MECC method for urine profiling. Cyclodextrin substitution, experimental conditions including capillary length, injection mode and time, applied voltage, temperature and capillary pre-conditioning procedures were investigated and optimised. Precision, linearity, sensitivity and robustness of the method were assessed, as well as urine stability. The validated sulphated β-cyclodextrin-modified MECC method allows for the separation of over 80 urinary analytes in under 25 min, using a sodium borate/SDS/sulfated β-cyclodextrin (25/75/6.25 mM) electrolyte and an 18 kV applied voltage in a 40 cm (effective length), 50 μm i.d. fused silica capillary at 20 °C, pre-conditioned with HCl for 5 min and BGE for 1 min, and UV diode array detection (190–600 nm). Such methodology should prove invaluable in the rapid comparison of urine profiles and indication of metabolic disorders or abnormalities.